The development and feasibility testing of a system capable of estimating velocity in small vessels at low flow velocities and mapping the vasculature in three dimensions is the central goal of this application. The ultimate objectives are to develop an ultrasonic technique for non-invasive assessment of blood velocity and vascular architecture capable of mapping the microvasculature and to apply this technique to assess subtle perfusion anomalies in ocular diseases such as glaucoma and retinal degeneration and to monitor the effects of pharmacological and surgical procedures used to treat these conditions. The proposed three dimensional (3D) processing has advantages both in the sensitive differentiation of slowly moving blood from the surrounding tissue, and in providing maps that allow a clinician to visualize the tortuous and complex vascular structure. Successful realization of these potentials would benefit other clinical applications including vascular examinations of the skin and lymphatic system. We will conduct the first high frequency study of flow though a small vessel tree, using 40-100 MHz transducers. Using a 38 MHz center frequency and new velocity estimation strategies we can detect and resolve vessels that are 40 micrometers in diameter or above and can distinguish velocities of 0.2 mm/s or above. With the extension to 100 MHz we expect to resolve vessels that are separated by 20 micrometers. While the sensitivity of commercial ultrasound systems to low velocity flow and small blood vessels has increased dramatically in the past few years, the spatial and velocity resolution have not been optimized to address this clinical application. The experimental systems used in our approach transmit a single cycle of a higher transducer center frequency, providing a significant improvement in spatial resolution. We use a technique called velocity-dependent signal processing with tracking of groups of red blood cells to produce low variance estimates of velocity over a wide range of velocity magnitudes. We have also developed a strategy for estimating the beam-vessel angle and 3D velocity magnitude, which is very useful for this clinical application. We will employ a strategy we have developed for using 3D velocity and amplitude to distinguish small vessels from tissue, based on the requirement for continuity of flow in a volume. Ultrasonic scanning of the eye has unique features due to the very limited depth of interest, allowing high center frequency, shorter signal, and use of 3D to improve our ability to reliably detect small blood vessels. In-vitro and in-vivo studies will compare the performance of this new system with results obtained with fluorescein angiography and color Doppler imaging.